To determine the associations between comorbidities, health-related quality of life (HRQoL) and functional impairment in people with mild-to-moderate chronic kidney disease (CKD) in primary care.Cross-sectional analysis at 5-year follow-up in a prospective cohort study.Thirty-two general practitioner surgeries in England.1008 participants with CKD stage 3  who survived to 5 years and had complete follow-up data for HRQoL and functional status (FS).HRQoL assessed using the 5-level EQ-5D version , and FS using the Karnofsky Performance Status scale . Comorbidity was defined by self-reported or doctor-diagnosed condition, disease-specific medication or blood result.Mean age was 75.8 years. The numbers reporting some problems in EQ-5D-5L domains were: 582 (57.7%) for mobility, 166 (16.5%) for self-care, 466 (46.2%) for usual activities, 712 (70.6%) for pain/discomfort and 319 (31.6%) for anxiety/depression. Only 191 (18.9%) reported no problems in any domain. HRQoL index values showed greater variation among those with lower FS  EQ-5D index value was 0.45 (0.24 to 0.68) compared with 0.94 (0.86 to 1) for those with Karnofsky score of 90). Overall, 234 (23.2%) had functional impairment.2) versus BMI <25 kg/m2: , self-care: OR 1.98 , usual activities: OR 1.82 , pain/discomfort: OR 2.37 ). Female sex, lower FS and lower educational attainment were independently associated with anxiety/depression , 3.08  and 1.67 , respectively). Older age, higher comorbidity count, albuminuria (≥30 mg/mmol vs <3 mg/mmol), lower educational attainment  and obesity were independently associated with functional impairment , 2.18 , 1.74 , 2.08  and 4.23 (95% CI 2.48 to 7.20), respectively).In multivariable logistic regression models, functional impairment was independently associated with experiencing problems for all EQ-5D-5L domains , p<0.001, usual activities: OR 8.27 (95% CI 5.43 to 12.58), p<0.001, pain/discomfort: OR 2.94 (95% CI 1.86 to 4.67), p<0.001, anxiety/depression: 3.08 (95% CI 2.23 to 4.27), p<0.001). Higher comorbidity count and obesity were independently associated with problems in mobility, self-care, usual activities and pain/discomfort: for three or more comorbidities versus none: , self-care: OR 2.64 , usual activities: OR 4.20 , pain/discomfort: OR 3.06 ), and for obese (body mass index (BMI) ≥30 kg/mThe majority of persons with mild-to-moderate CKD reported reductions in at least one HRQoL domain, which were independently associated with comorbidities, obesity and functional impairment.National Institute for Health Research Clinical Research Portfolio Study Number 6632. This study involved a large cohort of people with chronic kidney disease (CKD) recruited from primary care, a setting in which patients with mild-to-moderate CKD are typically managed in the UK.A broad range of comorbidities were included but they were identified at baseline only, not at follow-up, by which time the number of comorbidities may have changed.Health-related quality of life and functional status were measured in the same patient group and the use of the EQ-5D-5L index measure and data from the Health Survey for England enabled comparison with a general population.Health-related quality of life and functional status measures were taken at 5-year follow-up but not at baseline and we were therefore unable to identify change over time.This was a cross-sectional study of survivors and we are therefore not able to draw causative links.Chronic kidney disease (CKD) is common globally, affecting about 13% of the general adult population, with CKD stage 3 the most prevalent category.4Conversely, comorbidities  are common in individuals with CKD and can worsen clinical outcomes and health-related quality of life (HRQoL).6A significant body of research has explored HRQoL and the functional status (FS) of people with ESKD or following kidney transplant but these factors are not well explored in those with less severe CKD. Among 733 people with high-risk CKD in the Renal Impairment In Secondary Care study, 555 (76%) reported problems in one or more of the EQ5D domains.7 8This is a clinically important knowledge gap because mild-to-moderate reductions in glomerular filtration rate (GFR) are usually asymptomatic, so improved understanding of the comorbidities and symptoms that affect HRQoL and FS in this group of people is important to facilitate a holistic approach to management. The objective of this study was therefore to evaluate HRQoL and determine the associations between comorbidities, HRQoL and functional impairment in people with mild-to-moderate CKD in primary care.2, at least 90 days apart).A detailed description of the Renal Risk in Derby (RRID) study methodology has been published elsewhere.HRQoL was assessed at 5-year follow-up using the EQ-5D-5L, a widely used, validated measure of health status that can be standardised to different populations. EQ-5D-5L consists of two aspects: a descriptive system, in which participants are asked to rate their health state from 1 to 5 against five domains  and the EQ visual analogue scale (EQ-VAS), in which participants rate their health on a scale ranging from ‘the best health you can imagine’ 100) to ‘the worst health you can imagine’ (0).0 to ‘theFS, defined in this paper as the physical ability to perform normal activities and independently self-care, was assessed at 5-year follow-up using the Karnofsky Performance Status (KPS) scale. The KPS is a clinician-assessed score originally developed in oncology and was used for assessing prognosis and management in patients with cancer.16The methods for defining comorbidities in participants have been described in detail elsewhere.Kidney function was assessed at 5-year follow-up. eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation and was treated as a continuous variable. The urine albumin-to-creatinine ratio (uACR) from three consecutive early morning specimens was used for analysis. uACR was categorised into three levels according to KDIGO guidelines and fitted as a discrete variable in regression analyses.2 and uACR <3 mg/mmol at any study visit in an individual who had previously met KDIGO diagnostic criteria for CKD.Methods for defining CKD progression have also been detailed elsewhere.Body mass index (BMI) was calculated from weight in kilograms divided by square of height in metres and was treated as a categorical variable.2 test. Ratings from the five participant-reported EQ-5D-5L domains were also compared between the RRID cohort and those reported by people aged 65 years and over in the 2012 Health Survey for England (HSE)—which is representative of the England population.Descriptive statistics were used to show the characteristics of the study participants at 5-year follow-up. Descriptive statistics were also used to show the distribution of functional impairment (KPS ≤70) among those reporting problems in the five EQ-5D-5L domains. Associations between the patient-reported EQ-5D-5L domains and FS was assessed using the χUnivariable logistic regression models were used to assess the relationships between having ‘some problems’ in each EQ-5D-5L domain and each predictor variable, including comorbidity count and year-five eGFR. Variables considered to be clinically relevant and where p<0.1 on univariable analysis were subsequently included in multivariable logistic regression models. This process was then repeated for the relationship with the outcome variable functional impairment. Due to the small number of non-white participants, ethnicity was not included in the models.In the regression models, interactions between the individual and area measures of socioeconomic status were also tested because of the potential for the relationship between individual socioeconomic status  and HRQoL to vary by area deprivation, particularly for older people.https://www.uhdb.nhs.uk/renal-risk-in-derby-rrid-study/).The RRID study design was discussed with a patient and two feedback meetings for participants and their families were organised after the year 5 visits, which were well attended. In addition, a web page provides updates and information for participants  had complete 5-year follow-up data for HRQoL and FS . The mea2 (SD 15.2) and almost half  had had stable CKD over the preceding 5-year period. Only 56 (5.6%) had no comorbidities and about a third  had three or more comorbidities. For comparison of basic characteristics of those with and without complete 5-year follow-up data, see Approximately half  reported having had no formal education, just under half  lived in areas of lower deprivation (IMD quintiles four or five) and the majority  were white. The mean eGFR at follow-up was 54.0 mL/min/1.73 m10.1136/bmjopen-2020-040286.supp1Supplementary dataThe majority reported some impairment in HRQoL overall, with a median score of 75 out of 100 (IQR 60–90) on the EQ-VAS. A minority  had an EQ-5D-3L index score higher than the age/sex matched median, and only 18.9% of people (n=191) reported no problems across any of the individual HRQoL domains. Furthermore, a majority of participants reported some problems with mobility  and pain/discomfort  than in the HSE population .For clinician-assessed FS, only two participants had performance status assessed as KPS ≤40  and 232 (23%) were assessed as KPS 50–70 .The association between clinician-assessed FS and patient-reported HRQoL was complex, either when based on the index score  or the VUsing the mobility domain as an example , on univIn the fully adjusted multivariable model, these associations remained for older age, higher number of comorbidities, poorer FS and higher BMI . A summaFactors associated with a lower FS on univariable analysis included older age, lower socioeconomic status , higher number of comorbidities, obesity, reduced eGFR and greater degree of albuminuria. Other than reduced eGFR, all of these factors remained significant after adjustment . No inteIn this cross-sectional study of people with mild-to-moderate CKD who survived to year 5 in a UK primary care cohort, overall patient-reported HRQoL was relatively high though a substantial proportion of participants reported problems in each HRQoL domain. A majority reported problems with mobility and pain/discomfort. Although most people had a clinician-assessed FS suggesting that they were able to carry on normal activity and to work with no special care needed, about a quarter were assessed as having functional impairment . HRQoL was generally higher among those with better FS but there was more variation in HRQoL among those with lower FS, and low FS was independently strongly associated with low HRQoL in regression analyses. Higher number of comorbidities and obesity were independently associated with problems in most EQ-5D-5L domains and with functional impairment. Functional impairment was independently associated with experiencing some problems across all EQ-5D-5L domains.This study had several strengths, including the large size of the cohort, and recruitment from primary care, a setting in which patients with mild-to-moderate CKD are typically managed. The RRID cohort is pragmatic and likely to represent a population of typical patients with mild-to-moderate CKD in the UK.People with CKD are likely to have multiple comorbidities due both to the nature of the disease process and the relationship between CKD and older age. We have identified that comorbidity count was an independent determinant of both HRQoL and FS, highlighting the importance of a holistic approach that includes attention to comorbidities in the management of people with mild-to-moderate CKD. As reported previously, 40% of people in this cohort with stage 3 disease had at least two comorbidities.The prevalence of diabetes in this population of people with CKD stage 3 was 14.2%. This is lower than the prevalence of 20.1% noted in analyses of CKD prevalence in the HSE.2 was 23.6%.Mental health problems are common with 26% of adults in England reporting a diagnosis at some point in time.2) than people without, variously reported as between 26% and 55% depending on the nature of the population studied and the measure of ADL used.Being able to carry out activities of daily living (ADLs) is an important part of living independently. There is a recognised association between having difficulty with ADLs and a lower HRQoL in older adults, though comparison of HRQoL (as assessed by EQ-5D-5L) with FS (as assessed by the KPS) is understudied.In 2016, NICE published their first guideline for the management of individuals with multiple coexisting chronic diseases.We have observed that people with mild-to-moderate CKD commonly have multiple comorbidities and many report some HRQoL problems and functional impairment. Taken together with previous observations that the risk of CKD progression is low in this context, these data suggest that mild-to-moderate CKD in older people could be more important as a marker of increased comorbidity than as a risk factor for ESKD. Clinicians should therefore carefully consider comorbidities and discuss how treatments will fit in with those for other comorbidities, particularly mental health, chronic pain-related conditions and those affecting FS.


RNA was isolated from EVs and reverse transcription reaction was performed. Relative levels of miR-23a, miR-361, miR-1228 and miR-let7i were assessed in real-time qPCR using TaqMan probes. Analysis was based on the 2-ΔΔCT method. Statistically significant lower levels of miR-23a and miR-let7i were observed among NSCLC patients vs. control group: miR-23a, 0.054 vs. 0.107; miR-let7i, 0.193 vs. 0.369 . A receiver operating characteristic (ROC) curve analysis demonstrated the diagnostic potential of each individual serum EV-derived miRNA with an area under the curve AUC = 0.744 for miR-23a (p = 0.0003), 0.733 for miR-let7i (p = 0.0007). The decreased level of miR-23a in patients correlated with metastasis to lymph nodes and with AJCC tumor staging system. The results demonstrate that miR-23a and miR-let7i may prove clinically useful as significant, non-invasive markers in NSCLC diagnosis. Additionally, changing profile level of miR-23a that correlates with cancer development may be considered as an NSCLC progression marker.The aim of the study was a search for diagnostic and/or prognostic biomarkers in patients with non-small cell lung cancer (NSCLC) patients, based on circulating microRNAs  in extracellular vesicles (EVs). Serum EVs were isolated from NSCLC patients ( Lung cancer is the most common, aggressive malignancy, which is related to the highest number of deaths worldwide ,2,3. LunExperimental and clinical studies showed that qualitative composition of extracellular vesicles (EVs) may serve as reliable biomarkers and early diagnostic factors of cancer disease .The EVs MicroRNAs transported in EVs are highly conserved, non-coding, single-stranded, endogenous RNA molecules (21–25 nucleotides), that are transcribed by RNA polymerase II into pri-pre-miRNA . They caNumerous data show that miRNAs may act as oncogenes or tumor suppressors playing an important role in the regulation of the expression of genes involved in the carcinogenesis, metastasis and invasion, thus causing lung cancer reemission ,12,14. HThe expression of miRNAs: miR-23a, miR-361, miR-1228 and miR-let7i has been extensively studied in many types of human cancer (including lung cancer) and the significant dysregulation of their expression levels was observed in different biological materials isolated from patient ,26,27,28There are several studies on the role of miR-23 as an oncogene in various types of cancers, mainly due to its stimulatory effect on cell migration and invasion . MicroRNThe research conducted on the lung cancer cell lines showed that miR-1228 acts as a regulator of the tumorgenesis progress and cell response to environmental stress . SignifiDown-regulated expression of miR-361, which acts as tumor suppressor, is observed in gastric cancer tissues and cancer cell line models, colorectal cancer, prostate cancer, cutaneous squamous cell carcinoma and lung cancer ,25,31. IKRAS, c-MYC, CDK6, HOXA9, TGFBR1, BCL-XL, and MAP4K3) and key cell cycle regulators  [The let7 family is known to be a tumor suppressor, with significantly down-regulated expression observed in several cancers ,34. As fclin-D2) ,36,37. Tclin-D2) . Additioclin-D2) .In the light of many reports focused on evaluation of the circulating miRNA expression in patients with NSCLC, it seems reasonable to conduct further research in this area in order to improve therapy and diagnosis of NSCLC.The aim of the research is to specify the level of selected miRNAs in EVs: miR-23a, miR-361, miR-1228 and miR-let7i among NSCLC patients compared with health controls in order to search for NSCLC biomarkers, useful for diagnosis and monitoring of the disease.This study was conducted in accordance with Good Clinical Practice and the principles of the Helsinki Declaration. The protocols of this study were approved by the Bioethics Committee of the Medical University of Lodz (resolution No. RNN/85/18/KE of 13 March 2018). All participants signed an individual consent form for participation in the study.n = 31) patients with a confirmed diagnosis of NSCLC, 20 men and 11 women  were enrolled into the study. Patients were qualified in the Department of Thoracic Surgery, General and Oncologic Surgery, Military Medical Academy Memorial Teaching Hospital of Medical University of Lodz—Central Veterans’ Hospital, Lodz, Poland, between 2018 and 2019. The smoking status data was collected. A total of 14 patients were ex-smokers, 12 current smokers and 5 patients were never smokers. For further correlation analyses, smokers were divided into two groups: ≤40 PYs (n = 10) and >40 PYs (n = 13), depending on the numbers of pack-years  [A total of thirty-one (r Terms) . The resn = 21) consisted of age-matched  healthy female volunteers who participated in the screening tests at the Outpatient Clinic of Osteoporosis; Regional Center of Menopause and Osteoporosis, Military Medical Academy Memorial Teaching Hospital of Medical University of Lodz—Central Veterans’ Hospital, Lodz, Poland. The exclusion criteria included osteoporosis, diagnosis of cancer and the presence of other comorbidities that could significantly affect the study results.The control group .Regarding age, there were no statistically differences between NSCLC patients and control group , and serum was separated into new sterile tubes, frozen, and stored at −20 °C. Serum from all (n = 31) NSCLC patients was collected before the surgery. Serum from 21 patients (volunteers) was obtained as a control group, during a routine medical visit.A total of four milliliters of venous blood was collected into tubes without anticoagulant and left at room temperature until clot formation (about 30–60 min). Next, the samples were centrifuged  kit , according to the manufacturer’s protocol. Serum samples were thawed by placing them in a 25 °C water bath. To remove the cells and residues from the serum, samples were centrifuged at 2000× RNA isolation was performed using a Total Exosome RNA & Protein Isolation Kit , according to the manufacturer’s protocol.Qualitative and quantitative evaluation of the isolated RNA was performed by spectrophotometric method, measuring the absorbance with the Eppendorf BioPhotometerTM Plus , at 260/280 nm wavelengths. RNA with a 260/280 nm ratio in a range of 1.8–2.0 was considered to be of high quality and used to synthesize cDNA. Then RNA was aliquoted and frozen at −80 °C until the real-time polymerase chain reaction (qPCR) was performed.® MicroRNA Reverse Transcription Kit  and 3 μL of specific RT primers  included in individual TaqMan® MicroRNA Assays: hsa-miR-23a , hsa-miR-361 , hsa-miR-1228  and hsa-let7i  . Reverse Transcription (RT) master mix contained: 0.15 μL 25× dNTP Mix (100 mM), 1 μL MultiScribe™ Reverse Transcriptase (50 U/μL), 1.5 μL 10× RT buffer, 0.19 μL RNase Inhibitor (20 U/μL), and 4.16 μL nuclease-free water. Negative control was included in each RT reaction, containing no RNA (No RT control). RT reaction, in a total volume of 15 μL, was performed in a Personal Thermocycler . The RT reaction conditions were as follows: 30 min at 16 °C, 30 min at 42 °C, then 5 min at 85 °C and 4 °C hold. RT products were stored at −20 °C until further analysis.cDNA was transcribed from 5 μL (10 ng) of template RNA, using TaqMan® probes . A total reaction mixture volume of 20 μL contained: 1.33 μL RT Product, 1 μL TaqMan® MicroRNA Assays, 10 μL KAPA PROBE FAST qPCR Master Mix (2X) ABI Prism™ , and 7.67 μL nuclease-free water . The qPCR of miRNAs was performed on 96-well plates and assessed in the Applied Biosystems 7900 HT Fast Real-Time PCR System  for 39 cycles, with an annealing temperature of 60 °C; this was repeated three times for each sample.The levels of the studied miRNAs were assessed in real-time quantitative PCRs (qPCR) using TaqManMiRNAs level analysis was performed using DataAssist v3.01 , based on the global normalization method . The sofp < 0.05. The RQ values for the studied miRNAs are presented as mean ± standard deviation (median).The statistical analysis was carried out using the Statistica 13.1 program . The Shapiro–Wilk test showed that data were not normally distributed. In order to look for the statistical significance between the analyzed groups, Mann–Whitney U-test and/or Kruskal–Wallis tests were used, depending on the size of the groups. Neuman–Keuls’ multiple comparison test was used to identify possible significant differences in RQ values between the individual variables. The area under the receiver operating characteristic (ROC) curve was assessed to test the sensitivity and specificity of the studied miRNAs. The area under the curve (AUC) was resolved with a 95% confidence interval (CI). For all statistical analysis, the level of statistical significance was assumed at The qPCR results  for miRNAs were obtained for all NSCLC patients and control samples. For details, see p = 0.0030 and p = 0.0047, respectively, Mann-Whitney U-test) .The relative levels of the studied miRNAs were compared between NSCLC patients and controls. MiR-23a and miR-let7i were significantly down-regulated in NSCLC patients in comparison to control group (est) see . StatistReceiver operating characteristic (ROC) curve analyses were performed to calculate the diagnostic value of the studied miRNAs in NSCLC patients compared to healthy control group. A measure of the overall performance of a diagnostic test is the area under the ROC curve (AUC), which was calculated to estimate the specificity and sensitivity of miRNA to diagnose patients with NSCLC. The choice of an optimal cutoff point for dis-criminating between the NSCLC patients and the healthy controls was determined by the Youden index (J). J is the maximum vertical distance between the ROC curve and the diagonal reference line and is calculated as J = maximum (sensitivity + specificity − 1) .p = 0.0003) for miR-23a and 0.182 with a sensitivity of 71% and a specificity of 71% (p = 0.0007) for miR-let7i. AUC for miR-361 classifier was 0.538 and an optimal cutoff point was indicated at 0.57 with a sensitivity of 13% and a specificity of 0% (p > 0.05). AUC for miR-1228 classifier was 0.459 and an optimal cutoff point was indicated at 8.089 with a sensitivity of 81% and a specificity of 24% (p > 0.05)  see .p = 0.00001) . The highest AUC = 0.705 among the proposed panels was obtained for miR23a + miR-let7i. An optimal cutoff point for this panel was indicated at 0.076 with a sensitivity of 52% and a specificity of 83% (001) see . Combine001) see .The levels of miRNA were evaluated in correlation with such clinicopathological data as: age at time of diagnosis, sex and smoking history as well as histopathological characteristics of tumors (according to pTNM and AJCC classifications and NSCLC subtypes). For details, see p = 0.0313, Kruskal-Wallis test). Significantly lower miR-23a level was found in N2 than in N1   were noted (est) see .p = 0.0391, Kruskal-Wallis test). The Neuman–Keuls’ multiple comparison test found a borderline statistically significant decrease in miR-23a level in the stage III when compared to the stage II (p = 0.0696).According to the AJCC classification the relationship between the RQ values of miR-23a and the stage of tumor was revealed . Similarly, no associations were found with the histopathological NSCLC subtypes and tumor size (pT) according to the pTNM staging .Any statistically significant correlations were found between the RQ values of the studied miRNAs and the clinical features of NSCLC patients, i.e., patient age, sex, or history of smoking assessed as PYs  in the blood of patients with lung cancer . HoweverIn our research, due to the limited amount of material, we isolated EVs from human serum. The method is based on the polymer precipitation technique. The polymers bind water molecules, allowing the reagent to remove less soluble reagents from the solution and isolate them in the sediment by brief centrifugation. EVs after spin remain in pellets resuspended in PBS. The purity of the sample allows for further RNA isolation. The widely used approach isolation of exosomes from body fluids is based on ultracentrifugation in combination with sucrose density gradients or sucrose cushions to float the relatively low-density exosomes away from other vesicles and particles. These protocols can range in time from 8 to 30 h and a require large amount of material for successful isolation of exosomes . HoweverTumor cells secreting large amounts of EVs  seem to be essential from diagnostic and a prognostic point of view. In patients with diagnosed cancer, an isolated EVs fraction from blood is mainly composed of tumor derived particles . TherefoBased on the above reports, we analyzed the levels of three miRNAs, miR-26a, miR-29b and miR-519d in NSCLC tissue samples, and evaluated their diagnostic potential in NSCLC carcinogenesis . As a coIn the present study, we determined the profile of levels of four miRNAs , incorporated into blood circulating EVs and evaluated their potential role as diagnostic and/or prognostic biomarkers of NSCLC. We observed decreased levels of three out of four studied miRNAs  and an increased level of one miRNA (miR-1228) in NSCLC patients compared to reference patient. Significant differences were found for miR-23a and miR-let7i compared control group.The decreased level of miR-23a revealed in our study is in contrary to the results of other authors, who found its higher expression in plasma and different NSCLC cell lines ,21,51. HInterestingly, Kaplan–Meier meta-analysis of miR-23a expression level in various tissue cancers, including lung cancer, proved that its up-regulated level may be considered as a prognostic marker ,23. Sevelocus is often deleted, resulting in the reduced expression of this miRNA, and thus associated with poor prognosis [RAS and MYC oncogenes, direct targeting of DICER mRNA and cell proliferation control in a cyclin-dependent manner [RAS, c-MYC, CDK6, HOXA9, TGFBR1, BCL-XL, and MAP4K3) in the negative regulation of metastatic processes [RAS family and its level in cancer tissue is reversely correlated with cancer progression [An increasing number of publications confirms also the significance of miR-let7i and its correlation with the staging, differentiation, and metastasis of cancer . Thereforognosis . The putt manner . It was t manner . Furtherrocesses . MiR-letgression ,36,37,55gression .According to other authors, the modified miR-let7 expression was found in up to 50% of cells in lung adenocarcinoma cell lines and tissue samples, which led to increased cancer progression ,55. It wUsing the ROC curve analysis for miR-23a and miR-let7i separately, we found miR-23a to be more specific and miR-let7i more sensitive. The combination of miR-23a and miR-let7i shows greater specificity than miRNAs analyzed individually and similarly, moderate diagnostic value for NSCLC. Thus, these two miRNAs may have potential for further evaluation as biomarkers in the early diagnosis of NSCLC.In our study we evaluated EV-derived miRNAs levels, considering such factors as age at time of diagnosis, sex and smoking history. We did not notice any significant differences in the level of miR-23a and miR-let7i in relation to those parameters.Cigarette smoking is a significant risk factor for lung cancer. High expression of miR-23a in NSCLC tissues was shown to be significantly associated not only with the advanced stage of diseases but also independently with the smoking status. Current data suggests that smoking influences different molecular alterations, including dysregulation of miRNA expression level in tumor tissue, body fluids and serum in lung cancer ,61. In oOur study was limited by: small number of miRNAs assessed, small number of patients and no extra-cellular vesicles characterization.EVs as potential source of biomarkers are currently strongly considered, due to their easy accessibility and high stability. They contain information derived from the original tissues and as such, reflects this information using different molecules, including miRNA. Establishing of qualitative and quantitative molecular signatures of specific EVs is necessary in order to apply EV-based tumor diagnostic and cancer development monitoring.We confirmed the association between modified levels of EV-derived miRNAs and NSCLC as compared to healthy controls. Based on our data, we conclude that miR-23a and miR-let7i may be considered as potential non-invasive biomarkers differentiating healthy people from patients with diagnosed NSCLC. Nevertheless, this data requires further confirmation on a larger group of patients that would provide higher statistical significance.


Paralichthys olivaceus). RNA-seq was performed after challenge with Ringer’s at 8 h and 48 h. The data revealed that the expression profiles of blood, gill, and kidney were significantly changed. Differentially expressed genes (DEGs) were identified, and energy metabolic and immune-related genes were up-regulated or down-regulated obviously. GO and KEGG analyses showed that DEGs were mainly enriched in innate immune terms and pathways. Weighted gene co-expression networks analysis (WGCNA) also indicated the highest association module with stress. A total of 16 genes were detected in the gray module, which were immune-related and metabolic-related genes. These results provided fundamental information on intraperitoneal injection with solution buffer. It offered useful clues to further explore the functional mechanism of stress and immunity.Ringer’s or phosphate buffer saline (PBS) solution buffer usually was used as dilution butter in intraperitoneal injection. Stress could activate immune response, inflammatory response and glycogen metabolic process. The impact of solution buffer as a stressor to immune system was ignored in immunostimulatory experiment. In this report, we tested the hypothesis that the innate immune response and glycogen metabolic process were altered when it were challenged with Ringer’s in Japanese flounder ( Stress is a general term applying to a situation in which organism is subjected to a challenge . The strThe mechanism of these interactions has been mostly studied in higher vertebrates, but less information were available in fish. The studies focused on the regulation stimulated by high or low temperature. The normal expression profiles of kidney and gill were changed after challenged with high temperature in rainbow trout and Yesso scallop, and immune-related genes were up- and down-regulated . A serieRinger’s or PBS was usually used for immunostimulatory experiment as solution buffer to dilute bacteria. In fact, Ringer’s challenge was a stress, which could induce immune response. We should recognized it correctly, which might produce noise and interfere the result during immunostimulatory experiment. It could be recognized correctly, and exclude the noise caused by stress during immunostimulatory experiment. In our study, RNA-seq was used the estimate the influence of Ringer’s challenge at 8 h and 48 h post injection. Immune-related tissues were selected to detect the expression change after injection at transcriptome level. We demonstrated that the genes related to inflammatory response, innate immune, and metabolic process changed obviously. The results could provide a theoretical basis for the immunostimulatory experiment, especially intraperitoneal injection. The impact of solution buffer should not be ignored. It could be a reference for researchers to choose the better experimental method among injection, soaking, and feeding.All the experiments were conducted according to the Guidelines for the Institutional Animal Care and Use Committee of the Ocean University of China.The Japanese flounders used for the experiment were collected by the Yellow Sea Aquatic Product Co., Ltd., Shandong. A total of 12 healthy 1-year-old individuals were randomly selected for challenge experiment. The fish were divided into two groups denoted as the blank control group (BC group) and treatment group, which included four and eight individuals, respectively. During the experiment, the treatment group received an intraperitoneal injection of 1 mL sterilized Ringer’s solution buffer, and the BC group did not receive any treatment. The fish were maintained in sterilized seawater with sufficient oxygen after challenge. Before sampling, fish were anesthetized with MS-222. Four individuals were randomly collected from the BC group at 0 h post injection. At 8 h and 48 h post injection, four individuals were randomly collected from the treatment group. Tissues from blood, gill and kidney were collected and snap-frozen in liquid nitrogen and stored at −80°C. After collection, the samples were tested to exclude the infection of pathogen .Total RNA was extracted using TRIzol Reagent  in accordance with the manufacturer’s protocol, treated with RNase-free DNase I  to degrade genomic DNA, and then frozen at −80°C. RNAClean RNA Kit was applied to remove proteins. The quality and quantity were evaluated via 1.5% agarose gel electrophoresis and spectrophotometry using NanoPhotometer Pearl  and Agilent 2100 Bioanalyzer . The RNA-seq libraries of different tissues and time points were constructed by using Illumina TruSeq RNA Sample Prep Kit  in accordance with the manufacturer’s instruction. A total of 18 libraries were constructed, including BL-BC-1/2, BL-8 h-1/2, BL-48 h-1/2, G-BC-1/2, G-8 h-1/2, G-48 h-1/2, K-BC-1/2, K-8 h-1/2, and K-48 h-1/2. During the library construction, two biological replicates of each sample were used. Equal molar ratios of RNA from any two of four individuals were pooled as one replication at each time point in the same tissue. The remaining two individuals were pooled as another replication. Then the libraries were subjected to paired-end sequencing of 150 bp on the Illumina HiSeq 4000.Raw reads were cleaned by removing adaptors and low quality sequences using FastQC. TopHat was used to map the reads to the reference genome. Default values were set for the parameters of TopHat read mapping . Then thP < 0.01 were considered as DEGs. The DEGs were enriched by GO terms and KEGG categories using DAVID  and Kyoto encyclopedia of genes and genomes (KEGG) analyses were performed on DEGs at different time points in different tissues. The DEGs were significantly enriched in several GO terms in biological process, cellular component and molecular function levels. In blood, the terms involved in innate immune response, inflammatory response and immune system process were enrich significantly from 317 DEGs at 8 h post injection. Meanwhile, the terms related to immune response were enriched from 369 DEGs at 48 h post injection, including inflammatory response, innate immune response, defensing response to virus and TLR signaling pathway . In gillP < 0.05) (P < 0.05) (P < 0.05)  . In gill < 0.05) . In kidn < 0.05) . In thes < 0.05) . It was r2 = 0.86, P = 4e-06)  and obtained a system clustering tree of genes by gene-gene non-ω similarity . The hie= 4e-06) . The ME = 4e-06) . The selR = 0.7613, P < 0.01)  related with immune response were detected by qRT-PCR. Fold change was calculated by treatment group and control group. All the genes displayed consistent expression patterns both in qRT-PCR and FPKM ( < 0.01) .In conclusion, we demonstrated the change of expression profile post Ringer’s solution injection in Japanese flounder. The immune-related genes were up-regulated or down-regulated in different time points and different tissues. Besides, it was found that the genes involved in glycolysis/gluconeogenesis were also change after challenged with Ringer’s solution. These results indicated that the immune system and metabolic pathways were affected after Ringer’s solution challenge. This study revealed that Ringer’s solution buffer could influence immunostimulatory experiment during intraperitoneal injection.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Ocean University of China.JL and QZ conceived and designed the project, wrote and revised the manuscript. JL and ZL performed the experiments. JL and YW performed the data analysis. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lbx1, originate from somites and undergo extensive migration before becoming innervated by the hypoglossal nerve. How the complex form of HBM arose in evolution is relevant to the establishment of the vertebrate body plan, but despite having long been assumed to be similar to that of limb muscles, modification of developmental mechanisms of HBM remains enigmatic.Vertebrates are characterized by possession of hypobranchial muscles (HBMs). Cyclostomes, or modern jawless vertebrates, possess a rudimentary and superficial HBM lateral to the pharynx, whereas the HBM in jawed vertebrates is internalized and anteroposteriorly specified. Precursor cells of the HBM, marked by expression of Lbx genes in lamprey and hagfish (cyclostomes) and catshark . We show that the expression patterns of the single cyclostome Lbx homologue, Lbx-A, do not resemble the somitic expression of mammalian Lbx1. Disruption of Lbx-A revealed that LjLbx-A is required for the formation of both HBM and body wall muscles, likely due to the insufficient extension of precursor cells rather than to hindered muscle differentiation. Both homologues of Lbx in the catshark were expressed in the somitic muscle primordia, unlike in amniotes. During catshark embryogenesis, Lbx2 is expressed in the caudal HBM as well as in the abdominal rectus muscle, similar to lamprey Lbx-A, whereas Lbx1 marks the rostral HBM and pectoral fin muscle.Here we characterize the expression of Lbx genes would have been a prerequisite.We conclude that the vertebrate HBM primarily emerged as a specialized somatic muscle to cover the pharynx, and the anterior internalized HBM of the gnathostomes is likely a novelty added rostral to the cyclostome-like HBM, for which duplication and functionalization of  Many characteristics of vertebrate body plan are associated with the extremely complex morphology of skeletal muscles, as compared to the simple contractile tissues of invertebrate chordates, such as those found in amphioxus. Of the vertebrate skeletal muscles, hypobranchial muscles (HBMs), including tongue and infrahyoid muscles in jawed vertebrates, have attracted particular attention in evolutionary studies, due to their peculiar developmental processes. Precursor cells of the HBM originate from somites and migrate along an extensive, arch-shaped pathway circumventing the posterior margin of the posteriormost pharyngeal arch, as a cell stream called the hypoglossal cord . HBMs beLbx1, a member of lady bird class of homeobox transcription factor-encoding genes [Lbx1-deficient mice, the limb muscles are lost or severely affected, with tongue muscles reduced in size [Lbx2, a paralogue of Lbx1, has never been proposed to be involved in myogenesis in either tongue or limbs [The extensive migration of the somitic muscle precursors has been best studied in the formation of paired limbs, another feature shared by vertebrates. At the fore- and hindlimb levels, the ventrolateral lip (VLL) of the epithelial dermomyotome yields de-epithelialized, mesenchymal precursor cells for the limb muscles. The limb muscle precursors lose their original segmental configuration, forming a pool of mesenchymal progenitor cells that migrate distally into the limb buds, where they undergo muscle differentiation . These png genes , 5. In L in size . Howeveror limbs –9.In recent developmental studies, the cyclostome lampreys have served as an attractive model that retains the ancestral features of vertebrates –14. One Lbx gene, Lbx-A . In the Japanese lamprey Lethenteron camtschaticum (formerly Lethenteron japonicum), Lbx-A (LjLbx-A) mRNA has been shown to mark the anteriorly extending HBM precursors [LjLbx-A would retain the ancestral myogenic function of Lbx gene in HBM formation acquired early in vertebrate evolution. However, it remains unclear how Lbx1/Lbx2 genes of gnathostomes are functionally related to the single Lbx gene of the lamprey. In this study, we aimed to clarify how the duplicated Lbx genes are involved in the morphological complexity of gnathostome HBM by comparing myogenesis in the lamprey, hagfish, and shark.The lampreys, as well as the hagfish, the other group making up the cyclostomes, possess a single cyclostome-specific homologue of the ecursors . This obLbx-A gene during development. Since LjLbx-A sequence reported previously had lacked the 5′ part of the coding region [LjLbx-A utilizing the updated genomic data and the gene modeling  muscle, the posterior paired domain of HBM  and the lamprey HBM (associated with Lbx-A expression) are both attached with the ventral ends of pharyngeal muscles and do not fuse in the midline . The mRNA sequence of E. burgeri Lbx-A (LC506433) was predicted on the Ensemble Genome Database  and amplified from embryonic total RNA. We performed a search for genes of the catshark S. torazame  using the sequence archive Squalomix  containing the products of whole genome sequencing [Although we had previously reported partial mRNA sequence of LjLbx-A , we searLjLbx-A . A cDNA quencing . The corhttp://www.clustal.org) [Full-length amino acid sequences  . Positiotal.org) . The scatal.org) .To visualize differentiated muscles and neurons, we used the primary antibodies MF20 or A4.1025  and anti-acetylated tubulin . ZO-1  was used to detect tight junctions. The samples were subsequently reacted with the secondary antibody, the F(ab’) fragment of goat anti-mouse IgG (H + L) conjugated with horseradish peroxidase (Thermo Fisher Scientific). Sections were then counterstained with either hematoxylin or DAPI.LjLbx-A is highlighted in Additional file EbLbx-A was amplified with the primers 5′-CGCCCTGGAGGAACTCGCGAG-3′ and 5′-CATTGCCCTTAGCGTAAAGCF-3′. For the catshark, to distinguish the expression of Lbx1 and Lbx2, probes were designed to include the regions highly diverged between the two genes. Primers used to amplify the probe templates were Lbx1, 5′-GGACCTGGAGGAGATGAAGG-3′ and 5′-GACATGCGATGCAACAAGG-3′; Lbx2, 5′-CGAAGGGACTATTCCTTCTCT–3′ and 5′-AGTCCCACCCCTTCCCCAACAA-3′.Whole-mount and section in situ hybridization of lamprey, hagfish, and catshark embryos was performed as previously described , 41. In LjLbx-A locus  and guide RNA  dissolved in PBS. At appropriate stages, the posterior tail of each embryo was amputated and processed for genomic DNA extraction and PCR amplification for the LjLbx-A genomic locus by targeted PCR amplification followed by sequencing with Illumina MiSeq. We followed the previously described procedure [4, and 1.4 mM dNTP mix, followed by incubation at 95 °C for 1 min, 60 °C for 1 min, and snap-cooling on ice. Primer extension was performed by adding 1 μl (8 U) of the Bst DNA polymerase (NEB) to the reaction mixture, followed by incubation at 65 °C for 3 min and 80 °C for 20 min, and snap-cooling on ice. The combined PCR product, after the primer extension, was purified using AMPureXP beads, eluted in 10 μl of EB buffer (Qiagen), and further treated with 36 U of S1 nuclease (Takara) in a reaction mixture of 20 μl containing 1x S1 nuclease buffer at 23 °C for 15 min. The reaction was terminated by adding 0.5 μl of 0.5 M EDTA solution, and the DNA was purified using the AMPureXP beads and eluted in 10 μl of buffer EB. The combined PCR product after primer extension and digestion of the single-stranded region was analyzed by TapeStation (Agilent) to confirm the shortened length of the head and the barcode sequences (24 nt) before Illumina library preparation. Library preparation was performed as described previously [Each embryo was analyzed for the presence or absence of mutations in the rocedure  with modeviously  using 10https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The BBmerge function of BBTools ver. 37.88 [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). After separating the amplicon reads according to the barcode sequence for each individual  to obtain 250-nt-long paired-end reads. Quality control of the sequencing reads was performed using FastQC ver. 0.11.5 . Genomic structure of LjLbx-A gene and positions of CRISPR/Cas9 targets . Phylogenetic analysis of the cyclostome and catshark Lbx genes . Expression of LjLbx-A in the dorsal median fin muscle primordia . Workflow and additional data for lamprey genome editing experiments . Expression of catshark Lbx2 in the extending HBM precursor cells . ZO-1 staining of HBM primordium in the catshark embryo .Additional file 2: Table S1. Lbx genes compared in this study.Additional file 3: Table S2. Barcode primers used in lamprey genome editing experiments.


The N-Gh presents a much higher capacity than that of pure graphene prepared via the same method, which might be attributed to the introduction of nitrogen, which has much more effects and a disordered structure. As-prepared N-Gh exhibits a low O/C ratio that is helpful in maintaining high electrical conductivity. And the effects and disorder structure are also conductive to reduce the overlaps of graphene layers. A symmetric supercapacitor assembled with N-Gh electrodes displays a satisfactory rate behavior and long cycling stability .Doping heteroatom, an effective way to enhance the electrochemical performances of graphene, has received wide attention, especially related to nitrogen. Alternating voltage electrochemical exfoliation, as a low cost and green electrochemical approach, has been developed to construct  Because of this, it became one of the most significant candidate materials for next-generation electronic and energy storage devices  and hydroxyl (OH·) to open boundaries, which is helpful in facilitating 2 to expand the interlayer distance of graphite 2SO4) as anodic exfoliation electrolyte system shows a similar electrochemical mechanism in H2SO4 solution, except for the existence of OH− ions at the edge sites and grain boundaries  have been discussed to produce N-doped graphene in an anodic electrochemical exfoliation process  on a large scale by adding ammonium chloride salt to NaOH aqueous solution. Compared with as-prepared pure graphene (Gh) utilizing the same process, the N-Gh sample presents a larger size and much more effects. And the electrochemical properties of N-Gh have been investigated in three-electrode and two-electrode systems. The N-Gh sample reveals a satisfactory rate behavior and long cycling stability.In this study, the alternating voltage electrochemical technique has been successfully applied to 4Cl mixed aqueous solution. Both graphite rods were exfoliated via 5.0 V alternating voltage  for 5 h. Then the as-exfoliated substrate was separated, and further washed using distilled water until the pH value was close to 7. At last, the N-Gh sample was obtained via the freeze-dried method. Pure graphene (Gh) was also put in 3 M NaOH aqueous solution under the same preparation conditions.Alternating voltage electrochemical exfoliation was fabricated with a two-electrode system utilizing two graphite rods as working electrodes. N-doped graphene (N-Gh) was prepared in 3 M NaOH and 3 M NH+) from 10 to 80o at 5° min−1 with Cu Kα radiation. The raman spectra of the as-prepared products were collected using a Raman spectrometer (HORIBA Labram HR Evolution). The morphology of the as-obtained materials were explored through scanning electron microscopy  and transmission electron microscopy . Furthermore, the atomic arrangement was studied utilizing high-resolution transmission electron microscopy . Then, FT–IR spectrophotometer  was applied to test the surface functional groups of materials using KBr as a reference. Thermogravimetric analysis  from 25 to 900°C was utilized to measure the thermostability of materials with a heating rate of 5°C min−1 in air. Moreover, X-ray photoelectron Spectroscopy  was tested to analyze the surface chemical composition of the as-obtained samples with C1s photoelectron peak at 284.6 eV as the reference.The phase character of materials was studied via the X-ray diffractometer  curves were measured on MULTI AUTOLAB M204 (MAC90086) at various scanning rates. Electrochemical impedance measurements (EIS) were tested on a CHI 660B electrochemical working station with the frequency range between 100 and 0.01 Hz. Galvanostatic discharge/charge files were investigated at room temperature on Land CT2001A battery cycler.Active materials, super P, and polyvinylidene fluoride (PVDF) were mixed in N-methyl-2-pyrrolidone (NMP) with a mass ratio of 80:10:10 to form a coating slurry. Then, the as-obtained slurry was pressed onto a round nickel foam current collector. Finally, the electrodes with a loading mass of about 1.5 mg cm+ and OH− can intercalate into the graphite layers. Certain amounts of hydrogen gas can be produced during the electrochemical process, which would promote the exfoliation rate of graphite. For the N-Gh sample, the addition of − ions might be conducive to exfoliate the graphite electrode through much more intercalation. Moreover, the Cl− ions might be transformed into ClO− or Cl2 during the electrochemical anodic process . In The SEM has been utilized to analyze the morphologies of the as-prepared Gh and N-Gh, which is shown in −1 is related to E2g symmetry phonon mode, corresponding to ordered in-plane sp2 carbon atoms . This result again reveals that much more defects and disorder structure appear during the exfoliation process for N-Gh samples.−1 are related to O-H bending from H2O  which illustrate the existence of a few hydroxyl/phenolic/alkoxy groups on the surface of the exfoliated Gh and N-Gh samples  and oxygen carbons (C-O-C/C=O), respectively , respectively  , indicating the N-Gh//N-Gh symmetric supercapacitor might display good electrochemical reversibility. The charge/discharge files of N-Gh//N-Gh symmetric supercapacitor at the current densities between 0.5 and 10 A g−1 are revealed in −1 at current densities of 0.5, 1, 2, 5, and 10 A g−1, respectively. The energy density of this symmetric supercapacitor is 4.76 Wh kg−1 at a power density of 500 W kg−1, and the retention ratio is up to 68.7% at the power density of 10,000 W kg−1. Compared with some other N doped carbon-based materials  (Chang et al., To better study the electrochemical capacity performances of the as-obtained N-Gh, the symmetric supercapacitor has been fabricated with two N-Gh electrodes. in-situ alternating voltage electrochemical exfoliation technique with the introduction of NH4Cl in to NaOH aqueous solution. The N chemical states in the N-Gh sample mainly present pyrrolic nitrogen. Compared with the as-obtained pure Gh sample, the N-Gh shows a larger size, much more effects, and a disordered structure. Additionally, the related electrochemical behaviors have been investigated in a three-electrode aqueous solution system, indicating that N-Gh displays a much higher specific capacity than that of pure Gh. Moreover, it also displays a good cycling stability and high rate behavior with 63.7% of the capacity retention rate even at a current density of 20 A g−1. All these good electrochemical characteristics of N-Gh could be ascribed to the doping N, the existence of effects, and disorder structure, which is conducive to producing faradaic pseudocapacitance and reducing overlapping layers of graphene. The results of the symmetric supercapacitor fabricated with two N-Gh electrodes further illustrate the satisfactory cycling stability with 92.3% retention of N-Gh//N-Gh initial specific capacity after 5,000 cycles. These insights illustrate that the N-Gh sample prepared via an in-situ alternating voltage approach could have promising applications to construct composites for enhanced supercapacitors.In summary, the N-Gh sample has been prepared through an All datasets generated for this study are included in the article/supplementary material.MJ and TW designed and engineered the samples, performed the experiments, and wrote the paper. All authors contributed to performing the data analysis and general discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fucus distichus subsp. evanescens  on angiogenic and osteogenic processes in bone-related primary mono- and co-culture cell systems. By determining the gene expression and protein levels of the regulatory molecules vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG-1), ANG-2 and stromal-derived factor 1 (SDF-1), we show that the extracted fucoidans negatively influence angiogenic and osteogenic processes in both the mono- and co-culture systems. We demonstrate that purer fucoidan extracts with a high fucose and sulfate content show stronger effects on these processes. Immunocytochemistry of the co-culture system revealed that treatment with FE_F3, containing the highest fucose and sulfate content, impaired the formation of angiogenic tube-like structures, indicating the anti-angiogenic properties of the tested fucoidans. This study highlights how chemical properties of fucoidan influence its bioactivity in a bone-related context and discusses how the observed phenotypes can be explained on a molecular level—knowledge that is indispensable for future therapies based on fucoidans.Angiogenesis, the formation of new blood vessels from existing ones, is an essential process for successful bone regeneration. Further, angiogenesis is a key factor for the development of bone-related disorders like osteosarcoma or arthritis. Fucoidans, sulfated polysaccharides from brown algae, have been shown to affect angiogenesis as well as a series of other physiological processes including inflammation or infection. However, the chemical properties of fucoidan which define the biological activity vary tremendously, making a prediction of the bioactivity or the corresponding therapeutic effect difficult. In this study, we compare the effect of four chemically characterized high molecular weight fucoidan extracts from  Angiogenesis, the formation of new blood vessels from existing ones, is indispensable for bone regeneration and maintenance of bone health ,2,3. WheUndaria pinnatifida and Fucus vesiculosus were already “Generally Recognized As Safe (GRAS)” for the use in food by the Food and Drug Administration (FDA) in the USA. Additionally, the ionic nature of fucoidan and its biodegradability turn this marine molecule into a promising candidate for biomaterials in the field of tissue engineering and regenerative medicine [Fucoidan, a sulfated polysaccharide from the cell wall of brown algae and other marine invertebrates, has attracted increasing attention amongst researchers, because of the plethora of reported bioactivities. Fucoidans have shown to act on coagulation , angiogemedicine . Howevermedicine ,17. Varymedicine . InsuffiFucus distichus subsp. evanescens (FE) with a green and reproducible enzyme-assisted extraction. The extraction method uses enzymes that cleave specific cell wall components and release fucoidan in its native form [To better understand how fucoidans affect angio- and osteogenic processes on a molecular level in a bone tissue setting and what influence chemical properties have, we treated mono- and co-cultures of primary endothelial and osteoblast-like cells with four chemically well-characterized fucoidan extracts and quantified expression and protein levels of the important angiogenic regulators VEGF, SDF-1, ANG-1 and ANG-2. Additionally, we quantified alkaline phosphatase (ALP) activity and calcification level as early and late osteogenic differentiation markers, respectively. Therefore, we isolated human outgrowth endothelial cells (OEC) from blood  and meseive form . The cruThis paper (a) describes the bioactivity of four chemically well-characterized fucoidan extracts on angio- and osteogenesis in a mono- and co-culture system mimicking bone tissue, (b) provides information about the relationship between reported activities and chemical properties of the extracts, and (c) discusses a possible mechanism of action of HMW fucoidans in the context of angiogenesis in the mono- and co-culture systems.The fucoidans were isolated by an enzyme-assisted extraction method . The cru1H NMR revealed specific signals for fucose  in all fractions. A characteristic peak for uronic acids (glucuronic acid (GluA), ManA) however was only detected in the fraction FE_F1. The 1H NMR spectra gave indications for 1→3, as well as 1→4 glycosidic linkages. The spectra and further details can be found in the previous publication by Nguyen and colleagues [lleagues .The fucoidans in FE_crude have a molecular weight main peak around 400 kDa. The molecular weight of fucoidans in FE_F1 is more heterogeneous with the main peak around 400 kDa and with a smaller peak at 30–40 kDa. Both FE_crude and F1 have additional peaks around 2–3 kDa indicating alginate impurities. The molecular weight of the fucoidans in F2 ranges from 12–800 kDa with the main peak at 400–800 kDa and a shoulder around 40 kDa. Fucoidans in F3 have a more homogenous size with the main peak at 600 kDa.The sulfate content of the extracts increases with their purity. FE_crude, F1, F2 and F3 have a sulfate content of ~22, ~20, ~35 and ~39%, respectively.The total phenolic content of the extracts decreases with their purity. FE_crude, F1, F2 and F3 have a total phenolic content of 5.2, 2.9, 3.0 and 0.8 mgGAE per g dry weight, respectively.The total protein content is equal or less than 0.15% for all extracts and therefore negligible. The chemical properties of the tested extracts are displayed in To test the tolerance of OEC and MSC towards different doses of the enzymatically extracted fucoidans, we studied the metabolic activity and membrane integrity of the cells after seven days of treatment with five different fucoidan concentrations ranging from 1 to 200 µg/mL using MTS and LDH assays, respectively. Fucoidan treatment did not increase the release of LDH in both cell types, indicating that none of the extracts is cytotoxic a. The meTo examine how signaling molecules mediating angiogenic processes are influenced by the tested fucoidans in mono-culture and which role the chemical composition of fucoidan plays, we treated OEC and MSC for seven days with the extracts and quantified the expression and protein level of the angiogenic mediators VEGF, SDF-1, ANG-1 (MSC), and ANG-2 (OEC) in the respective mono-culture systems. The expression of VEGF and SDF-1 was downregulated after fucoidan treatment in MSC, while ANG-1 expression in MSC and ANG-2 expression in OEC was not affected a,b top. To assess whether osteogenic processes, which are tightly coupled to angiogenesis, were similarly impaired by the tested extracts, we quantified the expression and activity of the early osteogenic marker ALP after seven days and determined the calcification level as a late osteogenic differentiation marker after 14 days. Treatment with the purer fractions FE_F1, FE_F2 and FE_F3 decreased ALP expression almost completely a top. InTo visualize the influence of the fucoidan extracts on the formation of angiogenic tube-like structures, we co-cultured OEC and MSC and stained for the endothelial cell-specific adherens junction molecule VE-cadherin one week after fucoidan treatment. In the co-culture system, cells are able to interact, giving OEC the possibility to establish first angiogenic structures. Subsequently, we quantified the length and area of the emerged tube-like structures. Treatment with the fucoidan fractions FE_F1, F2 and F3 impaired the formation of these structures. The observed effect was strongest when cells were treated with the fraction FE_F3, richest in fucose and sulfates a. The quTo reveal more details on how fucoidans interfere with the angiogenic process on a molecular level, we determined expression and protein level of the angiogenic mediators VEGF, ANG-1, ANG-2 and SDF-1 in OEC-MSC co-culture after 7 days of treatment. In contrast to the mono-culture system, all the tested marker genes were expressed as in the control cells . While AF. evanescens. The extraction technique releases fucoidan likely preserving its native structural conformation. The obtained crude fucoidan molecules were fractionated by ion-exchange chromatography, also removing remaining sugar impurities like alginate oligosaccharides [In this study, we examined four HMW fucoidan extracts , differing mainly in monosaccharide content and sulfation degree, in regard to their effect on angio- and osteogenesis in primary OEC and MSC. Furthermore, we provide data to reveal details about the molecular mechanism that underlies the observed effects. OEC and MSC in mono- and co-culture were treated with four different fractions from enzyme-assisted extracted fucoidans from charides . Based oAngiogenesis is a very dynamic and carefully regulated process. To better understand the molecular mechanism underlying the observed anti-angiogenic phenotype caused by fucoidan treatment, we quantified VEGF, SDF-1, ANG-1 and ANG-2 expression and protein level in mono- and co-cultures .For the mono-culture system, we show that fucoidan downregulates VEGF and SDF-1 gene expression in MSC and decreases the protein levels of ANG-2 in the OEC and VEGF, SDF-1 and ANG-1 in the MSC cell culture supernatant . ResultsVarious studies already claimed that fucoidans interfere with the formation of blood vessels. However, due to different experimental set-ups and fucoidans, the results of these studies vary tremendously from anti- ,24,25,26F. evanescens, we quantified ALP expression and activity as an early and the degree of calcification as a late indicator for osteogenic differentiation. We found that the tested fucoidans negatively influence osteogenic processes in MSC mono-culture  and alginate lyase SALy from Sphingomonas sp. to break down non-fucoidan polysaccharide components of the cell and release fucoidan. High molecular weight alginate was removed by precipitating with CaCl2. Fucoidans were further precipitated using ethanol. The crude fucoidan product was lyophilized for bioactivity studies (FE_crude). The aqueous solution of the crude fucoidan was further purified and fractionated using ion-exchange chromatography, obtaining three fractions  as previously described in [Before the extraction, ribed in . Brieflyribed in . Subsequ2 gelatin method [1H NMR was performed with the Avance III-700 and with the Avance III-500 HD NMR spectrometer as specified in [The performance and detailed results of most chemical analyses of the fucoidan extracts are described in . Shortlyn method . Monosacified in .2CO3 were added and incubated for another 2 h. Subsequently, the absorbance was measured and the total phenolic content was expressed as gallic acid equivalents (GAE) in mg per g of dry fucoidan weight.The total phenolic content of the tested extracts was determined by the Folin–Ciocalteu method as described in . BrieflyThe total protein content of the tested extracts was determined with the Bradford assay using BSA as a standard .6 cells/cm2. After one week cells were detached by trypsination and sub-cultured in new collagen type I-coated 24-well plates at a density of 5 × 105 cells/cm2. Colonies of cobblestone-shaped OEC grew within two to three weeks. Obtained OEC were cultivated in fibronectin-coated  plates or flasks in EGM-2 including 7% FBS and 1% PS. The medium was exchanged every second day and cells were sub-cultured every three to four days when confluent.Human outgrowth endothelial cells (OEC) were isolated from human peripheral blood according to the previously published protocol . In brie6 cells/cm2. After the first sub-culture, the FBS content in the medium was reduced from 20% to 10%. Mesenchymal stem cells from passage number two were cultured in osteogenic differentiation medium , 10 mM β-glycerophosphate (Sigma-Aldrich) and 0.1 µM dexamethasone (Sigma-Aldrich)) to obtain osteoblast-like differentiated cells (MSC). The medium was exchanged every second day.Human mesenchymal stem cells derived from cancellous bone were isolated from femoral heads according to the previously published protocol . In brie2. On the next day, the culture medium was replaced by fresh medium containing the specific fucoidan concentrations. To keep the sampling time-point consistent for gene expression, protein level determination and immunocytochemistry staining, most experiments were harvested after seven days of treatment. For these experiments, the medium was refreshed on the third treatment day. Therefore, the old medium was replaced by fresh medium containing the same fucoidan concentrations as before. All experiments were performed with cells from three individual donors. Passage numbers of MSC and OEC ranged from 4–5.MSC or OEC were seeded in 96- or 24-well plates at a density of 40,000 cells/cmFucus vesiculosus from Sigma Aldrich (FV_crude (F5631) and FV_pure (F8190)) was included in the experiments.Control cells were cultured in medium without fucoidan. For a better comparability and as a reference substance, commercially available fucoidan from MSC or OEC were treated with 1, 10, 50, 100 or 200 µg/mL fucoidan. The LDH release as well as the metabolic activity of OEC and MSC were determined after one, three and seven days of fucoidan treatment. Pierce LDH Cytotoxicity Assay Kit  was used following the manufacturer’s protocol. The metabolic activity was assayed with the CellTiter 96 AQueous One Solution Cell Proliferation Assay  following the manufacturer’s protocol.TM PicoGreenTM dsDNA Assay Kit  in a 96-well plate. Therefore, 100 µL TE-buffer, followed by 72 µL PicoGreen reagent were added per well. Then, 28 µL sample or standard were added per well and incubated for 10 min at 200 rpm. The fluorescence was measured at 485 nm excitation and 535 nm emission wavelength.MSC were treated for seven days with 1, 10 and 100 µg/mL fucoidan. For harvesting, cells were washed with PBS and collected by scraping. The cell membrane was destroyed by freeze–thaw cycles and sonication. The released dsDNA content was quantified with the Quant-iTpnPP. Absorbance of the converted p-Nitrophenol was measured at 405 nm with a microplate reader after 1 h incubation at 25 °C.MSC were treated for seven days with fucoidan. The medium was collected and activity of ALP was measured using the Alkaline Phosphatase Assay Kit (Colorimetric)  according to the manufacturer’s protocol. In brief, samples were applied to a 96-well plate, followed by addition of ALP substrate MSC were treated for 14 days with fucoidan. Cells were fixed with 4% paraformaldehyde in PBS followed by the addition of 1 mL 40 mM Alizarin Red S Stain Solution (Millipore) to each well. After 30 min of incubation, cells were washed with distilled water until rinsed water became clear. To extract the bound dye, 600 µL 10% cetylpyridinium chloride (CPC)  was added to each well and incubated overnight. An amount of 150 µL CPC including the extracted dye was transferred to wells of a 96-well plate and the absorbance was measured at 560 nm. Samples were compared to a standard of alizarin red dilutions in CPC.2 on the first day. On the second day, OEC were seeded at the same density on top of the MSC. Cells were cultured in EGM-2 . On the next day, the culture medium was replaced by fresh EGM-2 containing the specific fucoidan concentrations. For cell experiments that were assayed or harvested after seven days, the medium was refreshed on the third treatment day. Therefore, the old medium was replaced by fresh medium containing the same fucoidan concentrations as before. All experiments were performed with cells from three different donors. Passage numbers of MSC and OEC ranged from 4 to 5 and from 4 to 8, respectively.MSC were seeded in fibronectin-coated plates at a density of 40,000 cells/cm® coverslips  in 24-well plates. After seven days of fucoidan treatment, angiogenic tube-like structures emerged and were stained for VE-cadherin. Therefore, cells were fixed with 4% PFA in PBS for 15 min, permeabilized with 0.5% TritonTM X-100 (Sigma-Aldrich) for 15 min and unspecific binding sites were blocked with 1% BSA in PBS for 30 min. Human VE-cadherin primary antibody (AF938 R&D) was applied in a concentration of 4 µg/mL in 1% BSA in PBS for 1 h after 3x washing with PBS for 5 min. AlexaFluor488-linked secondary antibody (A11055 Invitrogen) was applied in a concentration of 2 µg/mL in 1% BSA in PBS for 1 h after repeated washing. Nuclei were stained with Hoechst 33258 in a concentration of 2 µg/mL. Samples were mounted on objective slides using FluoromountTM (Sigma-Aldrich) and imaged with a fluorescence microscope.MSC and OEC were co-cultured on fibronectin-coated ThermanoxTM Kit  following the manufacturer’s protocol. After transcription, the volume was filled up to 100 µL with nuclease-free water and gene expression of VEGF, SDF-1, ANG-1, ANG-2 and ALP was determined with quantitative real-time PCR. Therefore, 3.2 µL cDNA was mixed with 16.8 µL master mix containing 10 µL SYBRTM Select Master Mix (Applied Biosystems), 2 µL QuantiTect Primer Assays , including DNA digestion via DNase I (VWR) treatment, according to the manufacturer’s protocol. An amount of 1 µg RNA was transcribed to cDNA using the High-Capacity RNA-to-cDNA® ELISA Development Systems  according to the manufacturer’s protocol.MSC/OEC mono- or co-cultures were treated for seven days with fucoidan. The medium was collected and protein levels of VEGF, ANG-1, ANG-2 and SDF-1 were quantified using DuoSetOEC-MSC co-cultures were treated with fucoidan for seven days. Cells were fixed and stained for VE-cadherin as described above. For each condition, three stitched microscopy pictures were taken, each consisting of nine single frames. Before quantifying the area and length of the angiogenic skeleton, the background of the microscopy pictures was subtracted, followed by a shading correction and frame stitching using the BASiC  and Gridp < 0.05.The mean values ± standard deviation from three independent experiments were plotted. Statistical significances were calculated with the Welch’s t-test or t-test using GraphPad Prism 7.03. Values were considered statistically significant when


The most important blood group system in cats is the AB, in which cats are classified as type-A, B or AB. Cats have antibodies against the blood type they do not possess, called alloantibodies. The aims of this study were to update blood type prevalence in cats from Northern Italy and study for the first time the blood type in cats from an insular region of Southern Italy, Sicily; to detect alloantibodies in these feline populations; to compare results with previous studies performed in Italy and between regions in Northern and Southern Italy. Cats from Southern Italy had the highest prevalence of type-B and type-AB, and the lowest prevalence of type-A blood in Italy. In particular, type-AB prevalence was higher than all previous reports in non-pedigree cats in Europe and the Italian prevalence of anti-type-B alloantibodies in type-A cats was the lowest reported worldwide. These results highlight the usefulness of regional studies to report different prevalences in feline blood types. Compatibility tests such as blood typing and cross matching must be considered fundamental in cats of any origin to ensure safe and efficient blood transfusion and to prevent neonatal isoerythrolysis.p = 0.0001) than in northern cats, while type-B and AB blood were significantly higher  in Southern compared to Northern Italian cats. Alloantibodies against type-A blood were found in 94.1% of type-B cats, 11.2% of type-A cats had alloantibodies against type-B blood, while no type-AB cats had alloantibodies with no significant difference between the two Italian populations. Type-AB prevalence in non-pedigree cats in Southern Italy was the highest reported in Europe. Italian type-A cats had the lowest worldwide prevalence of alloantibodies against type-B blood. These results highlight the usefulness of regional studies to report different prevalences in feline blood types and reinforce the importance of blood typing cats before transfusions and mating.The aims of this study were to determine the prevalence of A, B and AB blood types and alloantibodies in non-pedigree cats from two regions, one in Northern and one in Southern Italy . A total of 448 samples  were blood typed. The prevalence of A, B and AB blood types in northern and southern cats were 91.0%, 5.2%, 3.8%, and 77.2%, 12.1% and 10.7%, respectively. The prevalence of type-A blood in southern cats was significantly lower ( The most clinically important and well-studied blood group system in cats is the AB system in which cats are classified as types A, B or AB based on the molecular nature of the blood group antigen gangliosides ,2. Cats Type-A blood is the most prevalent blood type worldwide. Type-B and type-AB blood types were reported to be rare in initial studies of non-pedigree cats ,12,13,14Previous studies on blood type prevalence in cats from Italy, have investigated different populations of cats from Northern ,32 and CEthylenediamine tetra-acetic acid (EDTA)-anticoagulated surplus feline blood samples collected for various clinical reasons at the University of Milan  and at the University of Messina  were randomly included in the study between November 2014 and December 2015. Samples from all types of Italian cats were included in this study, i.e., samples collected from: (I) owned cats presented for routine check-ups before neutering, or for diagnostic purposes in the case of unhealthy cats, (II) shelter cats before surgery for neutering, (III) stray colony cats. This last group comprised stray cats that live in colonies in urban and rural areas of Italian cities and are captured for a trap, neuter, and release (TNR) sterilization program as part of a national program to control stray pet populations under Italian National Law (law no. 281/1991). Blood sampling in all cats was originally performed by veterinarians for the patient’s benefit and diagnostic purposes only and only surplus blood was used for this study. Based on both the University of Milan’s and the University of Messina’s animal use regulations, formal ethical approval was therefore not needed as cats were sampled for diagnostic purpose and informed consent was given by the owners, the director of the shelters and the legal representative of the feline colonies for use of surplus blood samples and data for scientific purposes. All analyses were performed at the Veterinary Transfusion Research Laboratory (REVLab) of the University of Milan where the Northern Italian cats were sampled. Blood samples from cats in Southern Italy were stored at 4–6 °C after collection and were shipped refrigerated to the REVLab where they were analyzed within 48 h of arrival. The sample size was estimated assuming a prevalence of 2.1% of type-AB cats based on results obtained in previous prevalence studies in the Northern Italian feline population . Therefog for 10 min at room temperature. Plasma was collected into a separate tube and was stored at −20 °C until tested for alloantibodies screening. The RBC pellet was washed by adding 2.5 mL of isotonic 0.9% NaCl solution. Following centrifugation at 1000× g for 1 min, the supernatant was removed, and the pellet was resuspended and then recentrifuged twice and finally reconstituted to a 5% RBC suspension. Polyclonal antibodies contained in type-B cat plasma  were used as primary reagents for the detection of type A red cell antigens. Triticum vulgaris lectin  was used for the detection of type-B RBCs antigens as this lectin binds to the NeuAc terminal of the type-B ganglioside and therefore strongly agglutinates feline type-B RBCs, but does not agglutinate type-A RBCs [® Tube Borosilicate glass 12 × 75 mm, Coming Inc., New York, NY, USA), 50 μL of 5% RBC suspension was mixed with 100 μL of type-B plasma , 100 μL of Triticum vulgaris lectin solution , or 100 μL of saline , respectively. These mixtures were incubated at room temperature for 15 min before centrifugation for 15 s at 1000× g. Tubes were then gently agitated, and blood type was recorded for the tube where macroscopic agglutination was present (Blood typing was performed using the tube agglutination technique (TUBE) as previously described ,36,37. B present . Type-B As samples collected in Sicily were shipped refrigerated to northern laboratory to be tested, we preliminarily assessed the stability of samples by typing one sample of each blood type, stored at 4 ± 2 °C, using TUBE after 24, 48, 72, and 96 h, and after 1, 2, 3, and 4 weeks of storage. Each sample stored at 4 ± 2 °C for up 1 month was correctly blood typed by TUBE.g. Agglutination was evaluated by gently agitating the tube to resuspend the non-agglutinating RBCs in the cell button. The presence of alloantibodies against RBCs of a different blood type was identified if macroscopic agglutination was present, as in the previous description of the tube technique for blood typing. In addition, where agglutination was not macroscopically visible, microscopic agglutination was evaluated at ×40x. Any RBC agglutination, macroscopic and/or microscopic, but not rouleaux presence alone, was considered a positive result for presence of alloantibodies in the tested plasma sample. Alloantibody screening is a modified major crossmatch, using RBCs of a known blood group, thus screening for naturally alloantibodies against that group in the recipient. Alloantibody screening was performed as previously described on frozen plasma samples ,39,40. BU test or Student’s t-test for independent samples were used. Comparisons between categorical data were made using Fisher’s exact test or χ2 test. Alloantibody prevalences between northern and southern cats were compared using comparison of proportions. A p value < 0.05 was considered significant. All statistical analyses were performed using a statistical software package .Prevalence of blood types and alloantibodies were calculated as the proportion of samples testing positive divided by the total number of tested samples and was presented with percentage and 95% confidence interval (95% CI). Data are shown as mean ± standard deviation (SD) or median and range (min-max) based on data distribution. To test the significance between age in Northern and Southern cats Mann–Whitney p < 0.0001). Sex was not reported in 20 cats  and no significant difference in male and female prevalence was found between northern  and southern  cats (p = 0.5025). A significantly higher number of owned cats were included in the northern (n = 120) population than from Southern Italy (n = 64) (p < 0.0001) and a higher number of colony stray cats were from Southern (n = 115) rather than from Northern Italy (n = 79) (p < 0.0001).A total of 448 blood samples were blood typed, 233/448 (52.0%) and 215/448 (48.0%) from the Lombardy region  and from Sicily , respectively. Samples were blood typed after a mean time of 12 days . Age was not recorded in 42 cats . The median age in Northern Italian cats  was significantly higher than in Southern ones   in non-pedigree DSH cats from Southern Italy and type-A less common (p = 0.0001) compared to cats from Northern Italy.Prevalence of blood types is reported in No statistical significant association was found between blood types and sex and origin  variables, either in the total feline population nor Northern and Southern Italian feline populations. Based on owner, shelter or colony postcode, cats with blood type-A, B or AB were not geographically clustered among the provinces from which cats were recruited both in Southern and in Northern Italy. The blood type prevalence in non-pedigree DSH cats previously reported in Italy and the results of the current study are reported in Alloantibodies screening were performed on frozen plasma samples within six months from RBCs separation. Due to insufficient residual volumes of plasma samples, alloantibodies were tested in only 327/448 (72.9%) samples: 196/233 (84.1%) from Northern Italy and 131/215 (60.9%) from Southern Italy . In partAlmost all  blood type-B samples had alloantibodies against type-A RBCs and all 32/32 (100.0%) showing a macroscopic agglutination reaction. Two type-B plasma samples did not agglutinate with type-A RBCs: a two month old owned DSH female kitten with panleukopenia from Northern Italy, and a 6 year old owned DSH male cat from Southern Italy with a wound on the chin. Only 30/268 (11.2%) type-A cats had alloantibodies against type-B RBCs, 13.4% (4/30) of these showed a macroscopic agglutination reaction while the majority  had only a microscopic agglutination. No type-AB tested samples (0/25) had alloantibodies against type-A or type-B RBCs. No significant differences in alloantibody presence were detected between southern and northern cats. This epidemiological study confirmed the prevalence of type-A blood at a worldwide high level in both investigated areas of Northern (Lombardy) and Southern (Sicily) Italy compared to studies in non-pedigree cats in Europe ,42,43,44The prevalence of blood type-B in non-pedigree cat populations in Europe varies from 0 to 30% ,41,42,49We highlight a high prevalence 10.7%) of type-AB cats in the studied region of Southern Italy, which is significantly higher than the 3.8% in cats from the region from Northern Italy. Prevalence of type-AB blood in previous Italian studies varied in non-pedigree cats from 2.1%  to 5.7% .7% of tyA alloantibodies or type-B samples were not tested with another method. This may explain variations in the prevalence of type-B and type-AB cats in the older epidemiological studies. There is less misclassification in more recent studies using the immunochromatographic method which has a higher sensitivity and specificity than the card-typing test for typing both type-B and AB samples [When considering the prevalence of blood type in cats, the blood typing method used must be considered. While TUBE is considered the gold standard technique in feline blood typing ,40, ques samples . To the best of our knowledge, this study is the first time sample stability has been tested for blood typing by TUBE. Based on our results, an additional advantage of TUBE is that the samples can be typed up to one month after collection (following storage at 4 ± 2 °C), and this could be useful in epidemiological studies and when working with samples collected from distant geographical regions, as in our study. Geographical variation has also been reported for alloantibody prevalence, and prevalence of anti-B alloantibodies in blood type-A non-pedigree cats ranged from 12.5% in Portugal  to 87.6%Two type-B cats had no alloantibodies. One was a 2-month-old kitten with panleukopenia. Usually type-B kittens develop their own anti-A alloantibodies at 6–8 weeks of age regardless of exposure to type-A erythrocytes. At 3 months of age, their plasma anti-A titers reach levels comparable with that found in adult type-B cats ,27,55. As expected, our study confirmed that type-AB cats do not have type-A or type-B alloantibodies as previously shown ,27,30.This study had a number of limitations. The most important limitation is the difference in populations between the samples from Northern and Southern Italy. The two feline populations were randomly sampled, but the cats from Northern Italy were older and more likely to be owned than those in Southern Italy, while more colony stray cats were blood typed from Southern Italy. However, these variables were not significantly associated with blood type or alloantibody prevalence ,47,49,54We provide for the first time information on the prevalence of blood type in cats from a region of Southern Italy, Sicily and of prevalence of alloantibodies in Italian cats. Cats from Southern Italy had the highest prevalence of type-B and type-AB and lowest prevalence of type-A blood in Italy. In particular, type-AB prevalence was higher than in all previous reports in non-pedigree cats in Europe and the prevalence of anti-type-B alloantibodies in type-A Italian cats was the lowest reported worldwide. These results highlight that regional studies are useful to report different prevalences in feline blood types and alloantibodies. Compatibility tests such as blood typing and cross matching must be considered fundamental in cats of any origin to ensure safe and efficient blood transfusion and to prevent neonatal isoerythrolysis.